West Nile (WN) and Japanese encephalitis (JE) viruses are flaviviruses that can cause a devastating acute neurological illness and currently there is no effective treatment for these viral infections. We have developed a RNA interference-based therapeutic method to suppress these viruses and shown the feasibility of using a single siRNA as a broad-spectrum antiviral agent to suppress fatal infections caused by both WNV and JEV in mice. However, the major challenge for harnessing the potential of siRNA for human therapy is the lack of suitable delivery methods. Delivery is particularly difficult in the CNS because of the presence of the blood-brain barrier. Preliminary studies suggest that a small peptide derived from the Rabies virus glycoprotein (RVG) may allow neuronal cell-specific targeting. Remarkably, when fused to a positively charged polymeric arginine peptide, the chimeric RVG-9R peptide was able to bind siRNA (by charge interaction) and deliver the siRNA to brain cells after intravenous injection in mice, resulting in specific gene silencing in the brain. Thus, we have identified a potential "siRNA drug" to treat flaviviral encephalitis as well as developed a clinically feasible method of delivering it to the brain by simple intravenous injection. In this proposal, in association with Alnylam Pharmaceuticals Inc, we will conduct preclinical evaluation of efficacy and safety, and advance the product development towards human therapy. Specifically, in aim1, we will conduct a comprehensive screen to select the most potent 2-3 lead antiviral siRNAs and chemically modify them for proper drug-like properties. In aim 2 we will address the bioavailability and safety/toxicology issues and determine the specificity of RNAi effects. In aim 3, we will test the efficacy of intravenous treatment to suppress encephalitis induced by JEV and WNV, both before and at different times after infection. To enhance the therapeutic index and safety of this treatment approach, in Aim 4 we will develop several different formulations of siRNA/peptide complex using RVG as the neuronal targeting agent. Relevance: These studies are expected to lead to the development of a viable RNAi-based treatment strategy for viral encephalitis. In addition, we would have developed a transvascular method for CNS delivery of siRNA and potentially other therapeutic agents that would facilitate treatment of a variety of neurological diseases.